This is a national stage application under 35 U.S.C. 371 of PCT/SE99/01434, filed on Dec. 10, 1999, now abandoned.
The present invention relates to a gene encoding a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster, and vectors and recombinant viruses containing said gene, as well as pharmaceutical compositions comprising such a vector and/or a virus. It also relates to production of said multisubstrate deoxyribonucleoside kinase and a process for phosphorylating nucleosides and nucleoside analogs.
Nucleoside analogs are commonly used in treatment of virus infections and cancer. The therapeutic nucleoside analogs are inactive prodrugs that are dependent on intracellular phosphorylation for pharmacological activity. The majority of nucleoside analogs in clinical use are phosphorylated by deoxyribonucleoside kinases (Arnxc3xa9r et al., (1995) Pharmac. Ther. 67, 155-186). These enzymes are intensively studied since they catalyze the rate limiting step in the pharmacological activation of the nucleoside analogs. There are four major deoxyribonucleoside kinases in human cells: deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) (1995) Pharmac. Ther. 67, 155-186). DCK, dGK and TK2 are closely sequence-related enzymes whereas TK1 has low similarity with the other deoxyribonucleoside kinases (Johansson et al., (1996) Proc. Natl. Acad. Sci. USA. 93, 7258-7262; Johansson et al., (1997) J. Biol. Chem. 272, 8454-8458; Chottiner et al., (1991) Proc. Natl. Acad. Sci. USA. 88, 1531-1535). The human deoxyribonucleoside kinases have distinct substrate specificities in regard to phosphorylation of both deoxyribonucleosides as well as nucleoside analogs.
WO95/14102 discloses recombinant adenoviruses comprising a DNA sequence coding for herpes simplex thymidine kinase under the control of a heterologous expression signal that can be associated with certain form of cancer. These recombinant viruses are then used to infect tumours of such a cancer. As a result, thymidine kinase is expressed in the cancer tumour. Subsequently, a therapeutic nucleoside analog prodrug, such as acyclovir (ACV, 9-(hydroxy ethoxymethyl)-guanine) and gancyclovir (GCV), is administred. Due to the enhanced expression of thymidine kinase in the tumor, the prodrug is only converted to the active form in the tumour, resulting in death of the tumour. However, the ability of thymidine kinase to phosphorylate potentially useful nucleoside analog prodrugs is limited.
WO97/29196 also relates to recombinant adenoviruses comprising a DNA sequence encoding herpes simplex thymidine kinase (HSV-TK). The kinase is mutated in order to increase the phosphorylation rate and to broaden the substrate specificity.
WO96/21724 discloses recombinant virus particles, such as recombinant retroviruses, contaning RNA encoding human deoxycytidine kinase 2. These virus particles are used for the same purposes as the virus particles described in WO97/29196 and WO95/14102, but the enzyme has another substrate specificity.
Accordingly, it is known to insert a xe2x80x9csuicidexe2x80x9d nucleic acid sequence, such as a nucleic acid sequence encoding a nucleoside kinase, into the genome of a virus or some other kind of vector capable of transferring nucleic acid sequences into tumour cells of a human or animal patient, and subsequently administer a therapeutic nucleoside analog prodrug. Known nucleoside kinases have a limited substrate specificity. HSV-TK, which is described in the above cited WO97/29196 and WO95/14102, cannot phosphorylate 2xe2x80x2,2xe2x80x2-difluorodeoxycytidine, 2-chloro-2xe2x80x2-deoxyadenosine, 1-xcex2-D-arabinofuranosylcytosine, 2xe2x80x2,3xe2x80x2-dideoxycytidine and 2xe2x80x2-deoxy-3-thiacytidine. Also deoxyguanosine kinase disclosed in the above cited WO96/2 1724 has been shown to have a limited substrate specificity. This enzyme does not phosphorylate), (E)-5-(2-bromovinyl)-2xe2x80x2-deoxyuridine, (E)-5-(2-bromovinyl)-1-xcex2-D-arabinofuranosyl-uracil, 2xe2x80x2,2xe2x80x2-difluorodeoxycytidine, 1-xcex2-D-arabinofuranosylcytosine, 2xe2x80x2,3xe2x80x2-dideoxycytidine or 3TC.
Consequently, there is a need for a DNA sequence encoding a nucleotide kinase having a broad specificity and a high catalytic rate of phosphozylation, in order to obtain flexibility regarding the use possible nucleoside analog prodrugs, and in order to reduce the dose amount required to obtain a sufficient therapeutic effect, resulting in a minimized risk for undesired side effects in the patient.
When comercially producing phosphoiylated nucleoside analogs., there is also a need for a nucleotide kinase having a broad specificity and a high catalytic rate of phosphorylation.
It has now turned out that by inserting a DNA or RNA sequence comprising a sub-sequence showing a homology of at least 60%, preferably at least 80%, and most preferably at least 90% of the DNA sequence of SEQ.ID.NO.1 into a cell, that cell will obtain a broad specificity for changing nucleoside analog prodrugs to active drugs by phosphorylation. Likewise this changement will occur at a high catalytic rate. Preferably the DNA sequence is inserted into the cell by transformation with a suitable virus or another suitable vector. Such viruses and vectors also constitute a part of the present invention.
A recent report (Munch-Petersen et al., (1998) J. Biol. Chem. 273, 3926-3931) shows that cell lines from the fruit fly Drosophila melanogaster contains only a single deoxyribonucleoside kinase. The report does neither reveal anything about the aminoacid sequence of the enzyme, nor about any DNA sequence encoding it. This enzyme, namned DM-dNK, is in contrast to the human deoxyribonucleoside kinases a multisubstrate enzyme. Although pyrimidine nucleosides are the preferred substrates of this enzyme, it catalyzes phosphorylation of both pyrimidine and purine deoxyribonucleosides. The enzyme also efficiently phosphorylates several anti-viral and anti-cancer nucleoside analogs. The catalytic rates of deoxyribonucleoside and nucleoside analog phosphorylation are, depending on the substrate, 10- to 100-folds higher than the maximal catalytic rates reported for the mammalian enzymes. The broad substrate specificity and high catalytic rate in phosphorylation of deoxyribonucleosides render DM-dNK unique among the family members of deoxyribonucleoside kinases.
Accordingly, an object of the present invention is to provide a nucleic acid sequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. Depending on the vector into which the nucleic acid sequence is intended to be inserted, the nucleic acid sequence can be a DNA sequence or a RNA sequence. The DNA may be cDNA, genomic DNA and synthetic DNA. It may also be double-stranded or single-stranded, and if single-stranded it may be the coding strand or the anti-sense strand. SEQ.ID.NO.1 discloses a cDNA sequence encoding the multisubstrate deoxyribonucleoside kinase. However, because of the fact that the genetic code is degenerated, other nucleic acid sequences encoding the same enzyme can be used in connection to the present invention.
Another object of the present invention is to provide a nucleic acid sequence comprising a disease-associated promoter and/or signal sequence operatively linked to a nucleic acid subsequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. Depending on the vector into which the nucleic acid sequence is intended to be inserted, the nucleic acid sequence can be a DNA sequence or a RNA sequence. It is also possible to inject an expression cassette comprising such a DNA sequence directly into cells that are to be killed.
Yet another object of the present invention is to provide a vector, such as a plasmid, cosmid or a bacteriophage, which vector contains a DNA sequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. Optionally, the vector also contains a disease-associated promoter and/or signal sequence operatively linked to the DNA sequence encoding a multisubstrate deoxyribonucleoside kinase. The invention also relates to host cells including these vectors.
Host cells are genetically engineered (transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes encoding the multisubstrate deoxyribonucleoside kinase. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent for the skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in anyone of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal (such as cDNA) and synthetic DNA sequences, e.g. derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequences may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli lac (lacI, lacZ) or trp. the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses, such as the promoters T3, T7, gpt, lambda PR, CMV immediate early, HSV thymidine kinase, early and late SV40 and late LTRs from retrovirus and mouse metallothionein-I. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. 
Yet another object of the present invention is to provide a process for producing a multisubstrate deoxyribonucleoside kinase by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells containing and capable of expressing a nucleic acid sequence showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2, under conditions promoting expression of said multisubstrate deoxyribonucleoside kinase and subsequent recovery of said multisubstrate deoxyribonucleoside kinase.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
Yet another object of the present invention is to provide a process for utilizing said multisubstiate deoxyribonucleoside kinase, or nucleic acid encoding such multisubstrate deoxyribonucleoside kinase, for example, to phosphorylate deoxyribonucleosides to ribonucleotides to activate specific anti-cancer and anti-viral drugs, and to preserve the fidelty of the deoxynucleotide pool.
Yet another object of the present invention is to provide a recombinant virus, such as a retrovirus or an adenovirus, whose genome comprises a disease-associated promoter and/or signal sequence operatively linked to a DNA sequence or a RNA sequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2.
Yet another object of the present invention is to provide a pharmaceutical comprising a recombinant virus, such as a retrovilus or an adenovirus, whose genome comprises a disease-associated promoter and/or signal sequence operatively linked to a DNA sequence or a RNA sequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2, together with a pharmaceutically acceptable carrier, excipient or diluent.
Yet another object of the present invention is to provide a conjugated multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2, which deoxyribonucleoside kinase is conjugated to a targeting group, such as a magnetic group or an antibody which specifically binds to an antigen associated with a disease, such as cancer or a virus infection.
Yet another object of the present invention is to provide a pharmaceutical composition comprising a conjugated multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2, which deoxyribonucleoside kinase is conjugated to a targetting group, such as a magnetic group or an antibody which specifically binds to an antigen associated with a disease, such as cancer or a virus infection, together with a pharmaceutically acceptable carrier, excipient or diluent.
As disclosed here, the term xe2x80x9cmultisubstrate deoxyribonucleoside kinasexe2x80x9d relates to a deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. The deoxyribonucleoside kinase is derived from Drosophila melanogaster and has a broad specificity for changing nucleoside analog prodrugs to active drugs by phosphorylation. Likewise this changement will occur at a high catalytic rate.
As disclosed herein, the terms xe2x80x9ctherapeutic nucleoside analog prodrugxe2x80x9d, xe2x80x9ctherapeutic nucleoside analogxe2x80x9d, and xe2x80x9cnucleoside analogxe2x80x9d, relates to nucleosides and analogs of nucleosides which are non-toxic and/or lack useful pharmaceutical characteristics, but which are transformed to potent pharmaceutically useful compounds when they are phosphorylated. Typically, they become cytotoxic after such a phosphorylation. Examples of such compounds include 9-(hydroxyethoxymethyl)-guanine (ACV), 1-xcex2-D-arabinofuranosyladenine (AraA), 1-xcex2-D-arabinofuranosylcytosine (AraC), 1-xcex2-D-arabinofuranosylguanine (AraG), 1-xcex2-D-arabinofuranosylthymine (AraT), 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxythymidine (AZT), 5-bromo-2xe2x80x2-deoxyuridine (BrdU), (E)-5-(2-bromovinyl)-2xe2x80x2-deoxyuridine (BVDU), 2-chloro-2xe2x80x2-deoxyadenosine (CdA), 2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxythymidine (D4T), 2xe2x80x2,3xe2x80x2-dideoxycytidine (ddC), dideoxythymidine (ddT), 2xe2x80x2,2xe2x80x2-difluorodeoxycytidine (dFdC), 1-(2-deoxy-2-fluoro-xcex2-D-arabinofuranosyl)-5-iodouracil (FIAU), 3xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxythymidine (FLT), (E)-5-(2-bromovinyl)-1-xcex2-D-arabinofuranosyl-uracil (BVaraU), 5-fluorodeoxyuridine (FdU), and 2xe2x80x2-deoxy-3-thiacytidine, 2xe2x80x2,2xe2x80x2-difluorodeoxyguanosine (dFdG), 2-fluoro-9-xcex2-D-arabinofuranosyladenine (FaraA), 5-aza-2xe2x80x2-deoxycytidine (5-AzadC), 5-fluoro-2xe2x80x2-deoxycytidine (5-FdC), 5-methyl-deoxycytidine (5-metdC), granciclovir (GCV), 1-(2-deoxy-2-fluoro-1-xcex2-D-abinofuranosyl)-5-thymine (FMAU) and 5-(2-bromovinyl)-2xe2x80x2-deoxycytidine BVDC).
As disclosed herein, the term xe2x80x9cdisease-associated promoter and/or signal sequencexe2x80x9d relates to a promoter or a signal sequence that is active in a cell affected by a disease, such as cancer or a virus infection. It is not a requirement that the promoter controls a gene that actively causes the disease, but it is a requirement that the gene that is controlled by the promoter is active in a cell affected by the disease. Preferably, the gene is not active in surrounding cells not affected by the disease. Likewise, it is not necessary that the signal sequence is directly involved in the mechanisms behind the disease. However, it is a requirement that the signal sequence actively targets the gene encoding the multifunctional nucleoside kinase to a cell affected by the disease. Examples are promoters and signal sequences originating from viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HIV), the promoter of the TK gene of herpes simplex virus type I, promoters of the adenovirus genes E1A, and MLP, the LTR promoter of Ross Sarcoma Virus, promoters of ubiquitous eucaryotic genes such as HPRT, PGK, alpha-actine, tubuline and DHFR, promoters from genes encoding filamentous proteins such as GFAP, desmine, vimentine, neurofilaments and keratine, promoters from therapeutically interesting genes such as MDR, CFTR, factor VIII, and ApoAI, promoters from genes that are specifically associated with certain tissues, such as the pyruvate kinase promoter, and promoters of intestinal fatty acid-binding proteins, promoters controlling the expression of oncogenes, etc. Signal sequences that can be used in relation to the present invention are nucleic acid sequences encoding a peptide sequence having the ability of directing the transport of a certain protein to the mitochondria (Zhu et al. (1998), J. Biol. Chem., vol. 273, 14707-14711; Johansson et al. (1996), Proc. Natl. Acad Sci. USA, vol. 93, 7258-7262) or the cell nucleus (Johansson et al. (1997), Proc. Natl. Acad. Sci. USA, vol. 94, 11941-11945).
The invention also relates to novel pharmaceutical and therapeutical agents which render it possible to specifically kill cells affected by a certain disease, such as cancer or a virus infection. Expression cassettes comprising a cDNA encoding the multisubstrate deoxynucleoside kinase of the present invention operatively linked to a disease-associated promoter or a signal sequence can be directly injected per se into the tissue to be treated. However, it is preferred to use some kind of vector to introduce DNA into the cells to be treated. Examples of such vectors are DEAE-dextran (Pagano et al., (1967) J. Virol. Vol. 1, p.891), nucleai proteins (Kaneda et al., (1989) Science, Vol. 243, p. 375), lipids (Felgner et al., (1987) Proc. Natl. Acad. Sci. USA, vol. 84, p. 7413) and liposomes (Fraley et al., (1980) J. Biol. Chem. Vol. 255p. 10431).
In a preferred embodiment of the present invention, the DNA or RNA sequence is transferred to the cells by using a recombinant virus comprising a nucleic acid encoding the multisubstrate deoxynucleoside kinase of the present invention as a vector. Examples of such virus are retroviruses (RSV, HMS, MMS, etc) and adenovirus.
The expression cassette or the virus comprising a nucleic acid sequence encoding the multisubstrate deoxynucleoside kinase according to the present invention can be formulated into pharmaceutical compositions suitable for various administration routes, such as topical, oral, parenteral, intranasal, intraveneous, intramuscular, intraveneous, subcutanous, intraocular and transdermal administration. Preferably, the pharmaceutical compositions are used in an injectable form. Accordingly, the pharmaceutical compositions comprise an expression cassette or a virus containing a nucleic acid sequence encoding the multisubstrate deoxynucleoside kinase according to the present invention together with a pharmaceutically acceptable vehicle which is suitable for an injectable solution which preferably can be injected directly into the tissue to be treated. Examples of formulations are sterile isotonic aqueous solutions, or dry, in particular lyophilized, compositions which can be transformed into injectable solutions by adding e.g. sterile water or serum.
According to the present invention, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administred to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a multisubstrate deoxynucleoside kinase of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be a retrovirus or an adenovirus which may be used to engineering cells in vivo after combination with a suitable delivery vehicle.
Once the multisubstrate deoxynucleoside kinase is being expressed intracellularly via gene therapy, it may be employed to treat malignancies, e.g. tumours, cancer, leukemias and lymphomas and viral infections, since the multisubstrate deoxynucleoside kinase catalyses the initial phosphorylation step of a therapeutic nucleoside analog prodrug. Examples of diseases that can be treated according to the principles outlined in the present application are: cancer in buccal cavity and pharynx, cancer in digestive organs (esophagus, stomach, small intestine, colon-rectum, liver, and biliary passages, pancreas), lung cancer, cancer of connective tissue, melanoma of skin, basal and squamous cell cancers, breast cancer, cancer in genital organs (cervix uteri, corpus uteri, ovary, prostate, testis), cancer in urinary organs (bladder, kidney), cancer of brain and central nervous system, cancer of endocrine glands (thyroid and other endocrine glands), leukemia and other cancers of blood and lymph tissues (Hodgkin""s disease, Non-Hodgkins""s lymphoma, multiple myeloma), human immunodeficiency virus (HIV) associated diseases, viral hepatitis, cytomegalovirus disease and other chronic infections caused by viruses.
A suitable dose of expression cassette or recombinant virus in relation to the present invention is a function of different parameters such as the vector/recombinant virus used, administration route, the particular pathology, or the duration of the treatment. A typical dose may be within the range from 109-1012 virus particles.
When the pharmaceutical composition comprising the expression cassette and/or the recombinant virus has been administred to suitable cells, these cells start to express the multisubstrate deoxyribonucleoside kinase. Then, a second pharmaceutical composition comprising a therapeutic nucleoside analog together with a pharmaceutically acceptable vehicle, excipient or diluent, preferably an injectable sterile aqueous solution is administred. The multisubstrate deoxyribonucleoside kinase according to the invention converts the therapeutic nucleoside analog to the active cytotoxic form resulting in cell death. A suitable dose of expression cassette or recombinant virus in relation to the present invention is a function of different parameters such as the vector/recombinant virus used, administration route, the particular pathology, or the duration of the treatment. A typical dose may be within the range of 100 mg-5000 mg.